The role of arginine methylation in regulating the function of
ribosomal S6 kinase 2

Khalil, M.I.M.;
(2010)
The role of arginine methylation in regulating the function of
ribosomal S6 kinase 2.
Doctoral thesis, UCL (University College London).

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Abstract

S6 kinases are serine/threonine protein kinases which play a key role in the control of
cell growth and metabolism. The S6K family has two members: S6K1 and S6K2. Both
isoforms are highly homologous in the kinase and kinase-extension domains and the
main divergence resides in non-catalytic regions which hint at differential functions
and/or regulation. Although much effort has been invested in finding S6K1 binding
partners/substrates, less attention has been given to the closely related S6K2. S6K2,
with the presence of a C-terminal NLS not found in S6K1 would indicates that S6K2
would has distinct nuclear functions. S6K2, unlike S6K1, is selectively recruited into a
signalling complex containing PKCε and B-Raf and likely controls FGF2-mediated
translation of mRNA species involved in the regulation of cell survival. In a previous
study in Ivan Gout’s laboratory, S6K2 was shown to bind directly to DNA via a short
motif at its extreme C-terminus, highly homologous to the AT-hook motif found in high
mobility group (HMG) DNA binding proteins.
To our knowledge, this study describes for the first time the modification of a protein
kinase by arginine methylation. This thesis details my attempt to understand the
functional importance of arginine methylation for S6K2 which is found to be at the
extreme C-terminus around the AT-hook motif. Here we have shown that S6K2 can be
methylated by Protein Arginine Methyltransferases (PRMTs). We demonstrate that
PRMTs methylates S6K2 primarily at Arg475 and Arg477 that are evolutionarily
conserved. Furthermore, the interaction between PRMTs (PRMT1, -3, and -6) and
S6K2 was demonstrated using a GST pull down, and revealed the importance of
AdoMet, the methyl donor, in enhancing the interaction of PRMT3 and -6 with S6K2.
The interaction was also confirmed by the co-immunoprecipitation assay indicating that
endogenous S6K2 forms a physical complex with endogenous PRMT1, -3, and -6 in
cells. In addition, S6K activity was found to be associated with PRMTs. Importantly,
the interaction between S6K2 and PRMT6 was found to be induced by serum
stimulation, while PRMT1 and PRMT6 knockdown reduces methylation of S6K2. One
interesting observation in this report, the specific demethylation of S6K2 upon
starvation of Hek293 cells which may imply the existence of specific demethylase(s) for
S6K2. We found that methylated S6K2 (wild type) found more in the nucleus than in
the cytoplasm, in other words, the mutants which lacks the methylation signal (R2M)
showed decreased nuclear distribution. Our results have clearly demonstrated that S6K2
mutant (R2M), lacking the methylation signal, promote serum starvation-induced
apoptosis when compared to the wild type and it seems that the methylation signal leads
to protection from apoptosis that follows growth factor(s) withdrawal. The molecular
mechanisms still need to be clarified. Taken together, we provide the first evidence that
arginine methylation serves as a regulatory modification for S6K2-mediated prosurvival
effect.

Type:

Thesis
(Doctoral)

Title:

The role of arginine methylation in regulating the function of
ribosomal S6 kinase 2